U.S. patent number 11,351,127 [Application Number 16/334,835] was granted by the patent office on 2022-06-07 for pharmaceutical composition.
This patent grant is currently assigned to Avexxin AS. The grantee listed for this patent is Avexxin AS. Invention is credited to Philip Hansen, Anders Ljungqvist.
United States Patent |
11,351,127 |
Hansen , et al. |
June 7, 2022 |
Pharmaceutical composition
Abstract
A pharmaceutical composition in the form of an emulsion, in
particular an oil-in-water emulsion, comprising certain
polyunsaturated long-chain ketones and polysorbate. The composition
may be used to treat or prevent certain inflammatory or
proliferative conditions.
Inventors: |
Hansen; Philip (Hosrsholm,
DK), Ljungqvist; Anders (Trondheim, NO) |
Applicant: |
Name |
City |
State |
Country |
Type |
Avexxin AS |
Trondheim |
N/A |
NO |
|
|
Assignee: |
Avexxin AS (Trondheim,
NO)
|
Family
ID: |
60009605 |
Appl.
No.: |
16/334,835 |
Filed: |
September 21, 2017 |
PCT
Filed: |
September 21, 2017 |
PCT No.: |
PCT/EP2017/073958 |
371(c)(1),(2),(4) Date: |
March 20, 2019 |
PCT
Pub. No.: |
WO2018/055062 |
PCT
Pub. Date: |
March 29, 2018 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20200261378 A1 |
Aug 20, 2020 |
|
Foreign Application Priority Data
|
|
|
|
|
Sep 21, 2016 [GB] |
|
|
1616093 |
May 24, 2017 [GB] |
|
|
1708329 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
3/10 (20180101); A61K 9/107 (20130101); A61K
9/0019 (20130101); A61K 31/121 (20130101); A61P
25/02 (20180101); A61K 47/26 (20130101); A61P
13/12 (20180101); A61P 29/00 (20180101); A61K
47/34 (20130101); A61K 47/14 (20130101); A61P
43/00 (20180101); A61P 19/02 (20180101) |
Current International
Class: |
A61K
31/121 (20060101); A61K 47/26 (20060101); A61K
47/14 (20170101); A61P 19/02 (20060101); A61K
47/34 (20170101) |
References Cited
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|
Primary Examiner: Hui; San Ming R
Attorney, Agent or Firm: McCarter & English, LLP Davis;
Steven G.
Claims
The invention claimed is:
1. A composition in the form of an aqueous emulsion comprising: (i)
1 to 20 mg/ml of a compound of the following formula: ##STR00007##
or a salt thereof; (ii) 1 to 20 mg/ml of a polysorbate surfactant;
(iii) 1 to 20 mg/ml of a triglyceride; and (iv) a buffer.
2. The composition of claim 1, comprising Polysorbate 80.
3. The composition of claim 1, comprising MCT oil.
4. The composition of claim 1, wherein the buffer is citrate
buffer.
5. The composition of claim 1, wherein the composition is
isotonic.
6. The composition of claim 1, wherein the composition has a pH of
about 6.0-9.0.
7. The composition of claim 1, comprising (i) 1 to 10 mg/ml of
compound A or B; (ii) 1 to 10 mg/ml of a polysorbate surfactant;
and (iii) 1 to 10 mg/ml of a triglyceride.
8. The composition of claim 1, further comprising a chelating
agent, an antioxidant or both.
9. An article of manufacture comprising a container holding the
composition of claim 1 and optionally, directions for using the
composition.
10. The article of manufacture of claim 9, further comprising an
inert gas for displacing air.
11. The composition of claim 1 in the form of an emulsion
comprising about 2 mg of compound B; about 2 mg of Polysorbate 80;
about 2 mg of MCT oil; and about 10 mmol of citrate buffer.
12. An article of manufacture comprising a container holding the
composition of claim 11 and optionally, directions for using the
formulation.
13. The composition of claim 1 in the form of an emulsion
comprising: from about 1 mg to about 500 mg of compound A or B
##STR00008## or a salt thereof; from about 0.1 mg to about 10 mg of
Polysorbate 80; from about 0.1 mg to about 10 mg of MCT oil; and a
buffering agent.
14. A method of treating or preventing an inflammatory or
proliferative condition comprising administering to a mammal, in
need thereof, an effective amount of a composition as defined in
claim 1.
15. The method of claim 14, comprising parenteral administration of
the composition.
16. The method of claim 14, comprising i.v., i.m., or s.c.
administration of the composition.
Description
This invention relates to a pharmaceutical composition in the form
of an emulsion, in particular an oil-in-water emulsion, comprising
certain polyunsaturated long-chain ketones. The invention also
relates to methods of treating or preventing certain inflammatory
or proliferative conditions using the composition of the
invention.
BACKGROUND
Certain polyunsaturated long chain ketones are described in various
prior art references for the treatment of conditions including
psoriasis, dermatitis, skin cancer, glomerulonephritis and
rheumatoid arthritis (See EP-A-1469859, WO2010/139482,
WO2012/028688, WO2014/082960 and WO2015/181135).
The polyunsaturated long chain ketones which are described in these
references have amphiphilic character but are primarily hydrophobic
and therefore insoluble in water. The lack of water solubility
limits the bioavailability of the compounds and limits the ability
of the skilled person to administer a useful dose of these
compounds to a patient. In particular, the lack of water solubility
limits the ability of the skilled person to administer the
compounds parenterally, e.g. intravenously, intramuscularly or
subcutaneously to a patient.
A further problem with the polyunsaturated ketone compounds of the
invention is that they are susceptible to degradation. Any
formulation of these compounds should also ensure that the
compounds remain stable for a prolonged period.
The present inventors sought a solution to the problem of
parenteral administration of these compounds and the problem of
their stability. The inventors have now found that these compounds
can be advantageously formulated as an emulsion, in particular an
oil-in-water emulsion. It is envisaged that the use of an emulsion
may lead to a prolonged shelf life and should also allow
administration of the compound of the invention by parenteral
routes such as intravenously (i.v.), intramuscularly (i.m.) and
sub-cutaneously (s.c.).
SUMMARY OF INVENTION
Thus, viewed from one aspect the invention provides a composition
in the form of an aqueous emulsion comprising:
(i) a compound of formula (I) R-L-CO--CF.sub.3 (I)
wherein R is an unsubstituted linear C.sub.10-24 unsaturated
hydrocarbon group, said hydrocarbon group comprising at least 4
non-conjugated double bonds;
L is a linking group forming a bridge of 2 to 5 atoms between the R
group and the carbonyl CO wherein L comprises at least one of S,
SO, SO.sub.2 in the backbone of the linking group;
or a salt thereof; (ii) a polysorbate and/or poloxamer surfactant;
(iii) optionally a triglyceride; and (iv) a buffer, chelating agent
and/or a lipid dissolvable antioxidant.
Viewed from another aspect the invention provides a composition in
the form of an oil-in-water emulsion comprising: (i) a compound of
formula (I) R-L-CO--CF.sub.3 (I)
wherein R is a linear C.sub.10-24 unsaturated hydrocarbon group,
said hydrocarbon group comprising at least 4 non-conjugated double
bonds;
L is a linking group forming a bridge of 2 to 5 atoms between the R
group and the carbonyl CO wherein L comprises at least one of S,
SO, SO.sub.2 in the backbone of the linking group;
or a salt thereof; (ii) a polysorbate and/or poloxamer surfactant;
(iii) a triglyceride; and (iv) a buffer and/or chelating agent.
Viewed from another aspect the invention provides a composition in
the form of an oil-in-water emulsion comprising: (i) a compound of
formula (I) R-L-CO--CF.sub.3 (I)
wherein R is a linear C.sub.10-24 unsaturated hydrocarbon group,
said hydrocarbon group comprising at least 4 non-conjugated double
bonds;
L is a linking group forming a bridge of 2 to 5 atoms between the R
group and the carbonyl CO wherein L comprises at least one of S,
SO, SO.sub.2 in the backbone of the linking group;
or a salt thereof; (ii) a polysorbate surfactant; (iii) a
triglyceride; and (iv) a buffer.
Viewed from another aspect the invention provides a method of
treating or preventing an inflammatory or proliferative condition
comprising administering to an animal, preferably a mammal, in need
thereof, e.g. human, an effective amount of a composition as
hereinbefore defined.
Viewed from another aspect the invention provides use of an
emulsion as hereinbefore described in the manufacture of a
medicament for use in the treatment or prevention of an
inflammatory or proliferative condition in an animal.
Viewed from another aspect the invention provides a composition as
hereinbefore described for use in the treatment or prevention of an
inflammatory or proliferative condition in an animal.
Viewed from another aspect the invention provides an article of
manufacture comprising a container carrying the composition as
hereinbefore defined.
Definitions
The term "compound of the invention" relates to an active agent of
formula (I), or a salt or solvate thereof, in particular a compound
A or B as herein defined.
The terms "oil-in-water formulation" and "oil-in-water emulsion"
are used interchangeably to describe a dispersion in an aqueous
phase. The "dispersed phase" comprises the compound of the
invention in an aqueous phase (the "continuous phase").
The term "nanoemulsion" relates to an oil-in-water emulsion in
which the dispersed oil phase comprises "nanodroplets" which are
hydrophobic droplets having a diameter of nanometers, i.e. from 1
to under 1000 nm, in a continuous aqueous phase.
DETAILED DESCRIPTION
This invention concerns emulsions, especially oil-in-water
emulsions, comprising compounds of formula (I) or salts thereof and
their use in the treatment or prevention of various conditions such
as inflammatory or proliferative conditions. The compound of the
invention is preferably present in the dispersed phase of the
oil-in-water emulsion.
Compounds of the Invention
The emulsion comprises at least one compound of formula (I):
R-L-CO--CF.sub.3 (I)
Preferably, only one compound of formula (I) is present in the
emulsions of the invention.
The group R preferably comprises 5 to 9 double bonds, preferably 5
to 8 double bonds, e.g. 5 to 7 double bonds such as 5 or 6 double
bonds. These bonds should be non-conjugated. It is also preferred
if the double bonds do not conjugate with the carbonyl
functionality.
The double bonds present in the group R may be in the cis or trans
configuration however, it is preferred if the majority of the
double bonds present (i.e. at least 50%) are in the cis
configuration. In further advantageous embodiments all the double
bonds in the group R are in the cis configuration or all double
bonds are in the cis configuration except the double bond nearest
the carbonyl group which may be in the trans configuration.
The group R may have between 10 and 24 carbon atoms, preferably 17
to 19 carbon atoms.
The R group is unsubstituted. The R group is linear. It preferably
derives from a natural source such as a long chain fatty acid or
ester.
The linking group L provides a bridging group of 2 to 5 backbone
atoms, preferably 2 to 4 backbone atoms between the R group and the
carbonyl. The atoms in the backbone of the linker may be carbon and
heteroatoms but will include at least one of S, SO, or SO.sub.2.
The linking group is preferably unsubstituted. It is preferably
linear.
Preferred components of the linking group are --CH.sub.2--, --S--,
--SO--, and --SO.sub.2-- which can be combined with each other in
any (chemically meaningful) order to form the linking group. Thus,
by using two methylene groups and an --S-- group the linker
--SCH.sub.2CH.sub.2-- is formed.
The linking group L contains at least one heteroatom in the
backbone. It is also preferred if the first backbone atom of the
linking group attached to the R group is a heteroatom or group of
heteroatoms selected from --S--, --SO--, and --SO.sub.2.
It is highly preferred if the linking group L contains at least one
--CH.sub.2-- link in the backbone. Ideally the atoms of the linking
group adjacent the carbonyl are --CH.sub.2--.
It is preferred if a heteroatom --S--, --SO-- or --SO.sub.2 is
positioned .alpha., .beta., .gamma., or .delta. to the carbonyl,
preferably .beta. or .gamma. to the carbonyl.
Highly preferred linking groups therefore are --SCH.sub.2--,
--SOCH.sub.2--, or --SO.sub.2CH.sub.2--
Preferred compounds of formula (I) are those of formula (I')
R--Y1-CH.sub.2--CO--CF.sub.3 (I')
wherein R is as hereinbefore defined; and
Y1 is selected from S, SO or SO.sub.2.
Highly preferred compounds for use in the invention are depicted
below.
##STR00001##
where X is CF.sub.3.
The following compounds, especially compound B, are highly
preferred for use in the invention:
##STR00002##
Where possible, the compounds can be present in the emulsion as a
salt or solvate. Preferably however, no such form is used.
Compounds of formula (I) may be manufactured using known chemical
synthetic routes found in J. Chem. Soc., Perkin Trans 1, 2000,
2271-2276 or J. Immunol., 1998, 161, 3421.
It has now surprisingly been established that compounds having the
structures described herein can be formulated as an emulsion, such
as an oil-in-water emulsion, preferably a nanoemulsion, when
combined with a polysorbate or poloxamer surfactant and optionally
a triglyceride excipient. It has surprisingly been found that
emulsions of the compounds described have excellent long term
storage stability, particularly at refrigerator or freezer
temperatures.
Without wishing to be bound by theory, the compounds described have
amphiphilic character but are primarily hydrophobic. It is thought
that as a result of these characteristics, the compounds of the
invention are capable of forming ordered nanostructures, such as
micelles, in an aqueous environment. These micelles are not a
structure which would usually be adopted by the compounds in pure
form or in conventional solvents. The inventors have now
established that particularly stable structures, such as mixed
micelles, comprising both the compound of the invention and the
triglyceride excipient have excellent storage stability.
The amount of compound of the invention in the emulsion can vary.
Suitable amounts include 1 to 500 mg, such as 1 to 300 mg, more
preferably 1 to 250 mg, especially 1 to 150 mg, especially 1 to 100
mg per ml of emulsion. Values of 1 to 20 mg/ml, such as 1 to 10
mg/ml are especially preferred.
Polysorbate and/or Poloxamer Surfactant
The emulsions of the invention include a polysorbate and/or
poloxamer surfactant. Appropriate levels of surfactant are 0.1 to
100 mg, such as 0.1 to 25 mg of surfactant in 1 mL of emulsion,
preferably 0.2 to 20 mg, such as 0.5 to 15 mg, and especially 1 to
12 mg per mL of emulsion.
Polysorbate surfactants are based on sorbitan, which is a sugar
derivative having four OH groups per molecule. Suitable surfactants
are those in which the OH groups of sorbitan are capped with
repeating ethoxy groups and one of the repeating ethoxy groups is
itself capped with a fatty acid ester.
Polysorbates such as polysorbate 20, 40, 60 or 80 could be used. A
particularly preferred surfactant is Polysorbate 80.
Alternatively, the surfactant may be a poloxamer. The term
"poloxamer" is a term of the art and describes block polymers
having a central block of polyoxypropylene and two outer blocks of
polyoxyethylene. A particularly preferred poloxamer is Poloxamer
188.
In general, polyethoxylated non-ionic surfactants are suitable
herein.
It is possible to use a mixture of surfactants such as two or more
polysorbates, two or more poloxamers or a poloxamer and a
polysorbate. Preferably, one surfactant is used.
In a preferred embodiment, the active compound is compound A or B
defined above and the surfactant is a polysorbate, especially
polysorbate 80.
Triglyceride Excipient
In addition to the surfactant, compositions of the invention can
comprise one or more triglycerides, preferably one triglyceride.
The triglyceride component ideally acts to dissolve the compound of
the invention to encourage formation of micelles in an aqueous
environment in the presence of the surfactant.
Where the triglyceride forms a minor component, it can be seen
assisting in the formation of micelles and/or improving the
stability of micelles formed by the compound of the invention. In
the event that the excipient forms a major component (i.e. is
present in a greater amount percentage by weight than the compound)
it can be seen as a type of carrier for the compound.
The triglyceride is preferably a medium chain triglyceride (MCT),
which are compounds having a glycerol backbone esterified with
three C6-C12 fatty acids, especially caproic acid (C6:0), caprylic
acid (C8:0), capric acid (C10:0) or lauric acid (C12:0). Suitable
sources include corn oil, sunflower oil, peanut oil, olive oil,
coconut oil, or most preferably MCT oil, which is a highly purified
coconut oil.
Suitable levels of excipient are 0.1 to 500 mg, such as 0.1 to 25
mg of triglyceride in 1 mL of emulsion, preferably 0.2 to 20 mg,
such as 0.5 to 15 mg, and especially 1 to 12 mg per mL of emulsion.
Where mixtures of triglycerides are used as the excipient, these
levels relate to the mixture of triglycerides.
In a preferred embodiment the compound is compound A or B as herein
defined and the triglyceride is MCT oil. In a yet even more
preferred embodiment the compound is A or B, the triglyceride is
MCT oil, and the surfactant is Polysorbate 80.
In a preferred embodiment, there can be
(i) 1 to 20 mg/ml of a compound of formula (I) as hereinbefore
described;
(ii) 1 to 20 mg/ml of a polysorbate and/or poloxamer surfactant;
and optionally
(iii) 1 to 20 mg/ml of a triglyceride.
In a preferred embodiment, there can be
(i) 1 to 10 mg/ml of a compound of formula (I) as hereinbefore
described;
(ii) 1 to 10 mg/ml of a polysorbate and/or poloxamer surfactant,
e.g. polysorbate 80; and
(iii) 1 to 10 mg/ml of a triglyceride, e.g. MCT oil.
Ideally the balance of the emulsion is the buffer and/or chelating
agent (and water).
Other Components
The emulsion of the invention includes a buffer, a lipid soluble
antioxidant and/or chelating agent. Preferably, the emulsion of the
invention comprises a buffer and/or a chelating agent. Ideally, the
emulsion contains a buffer.
Ideally any buffer present will maintain the pH of the emulsion at
around 7. Preferably the buffer maintains the pH of the emulsion in
the range of 6 to 9, preferably 6 to 8, more preferably pH
7.+-.0.5. Any suitable buffer may be used. However, a particularly
preferred buffer is citrate buffer. Ideally, the emulsion is
isotonic. The molarity of the buffer may be 5 to 20 mmol, such as
10 mmol.
Certain buffers such as citrate buffer may also bind traces of
metals and thereby reduce oxidative degradation of the compound of
the invention.
Alternatively or as well as the buffer, a separate chelating agent,
e.g. EDTA or a salt thereof, may also be present.
The compositions may also comprise a lipid dissolvable antioxidant
such as tocopherol or other vitamin E analogue.
The emulsions of the invention may also contain other active
components, e.g. other drugs, although this is not preferred.
In a most preferred embodiment, the emulsion consists of the
compound of the invention, the polysorbate or poloxamer surfactant,
the triglyceride excipient and a buffer and/or chelating agent
(along with water).
In a further preferred embodiment, the invention provides a
pharmaceutical composition in the form of an aqueous emulsion
comprising from about 1 mg to about 500 mg of a compound of formula
A or B
##STR00003## where X is CF.sub.3;
about 0.1 mg to about 10 mg of Polysorbate 80,
about 0.1 mg to about 10 mg MCT oil; and
a buffering agent, e.g. a citrate buffer.
Nanoemulsions
In a preferred embodiment, the emulsion of the invention is a
nanoemulsion. The nanoemulsion comprises a dispersed oil phase and
a continuous aqueous phase. The particles of the dispersed phase
have an average particle size of from 1 to less than 1000 nm,
preferably 5 to 200 nm. Particle size can be measured by routine
means, e.g. by microscopy (TEM, SEM, AFM), light scattering
techniques (Photon Correlation Spectroscopy (PCS)), etc.
The emulsions of the invention, such as the nanoemulsions of the
invention, are generally formed by mixing the components (compound,
surfactant, excipient and any additives) and making the volume up a
predetermined level using water for injection (WFI). It may be that
the compound of the invention is precontacted with the triglyceride
before addition of the surfactant. The water will typically be
added containing the buffer/chelating agent.
The mixture of components can then be agitated, e.g. using a shaker
and/or with sonication. The emulsion may be sterile filtered after
production.
Stability
The compounds present in compositions of the invention can
decompose into a variety of by-products. In stability studies, the
inventors have determined that particularly notable by-products are
sulfoxide and thiol compounds derived from the compound of the
invention. The generation of by-products can be reduced by
formulation of the compounds as an emulsion as described
herein.
As is illustrated by the examples, emulsions comprising a
surfactant and an excipient of the invention have excellent long
term storage stability. By "stable" it is meant that the compound
peak area, as measured by chromatograph, is reduced by no more than
20% after 1 month of storage at 5.degree. C., and preferably no
more than 20% reduction after 3 months of storage at 5.degree. C.,
preferably no more than 20% reduction after 6 months of storage at
5.degree. C. in an inert atmosphere.
Preferably the peak area is reduced by no more than 10% after 1
month, 3 months or 6 months of storage at 5.degree. C. Most
preferably the peak area is reduced by no more than 5% after 1
month, 3 months or 6 months of storage at 5.degree. C. in an inert
atmosphere.
Preferably the compound peak area, as measured by chromatograph, is
reduced by no more than 5% after 1 month of storage at -20.degree.
C., and preferably no more than 5% reduction after 3 months of
storage at -20.degree. C., preferably after 6 months of storage at
-20.degree. C. in an inert atmosphere. Preferably the peak area is
reduced by no more than 3% after 1 month, 3 months or 6 months of
storage at -20.degree. C. in an inert atmosphere. Most preferably
the peak area is reduced by no more than 2% after 1 month, 3 months
or 6 months of storage at -20.degree. C. in an inert
atmosphere.
Article
The compositions of the invention are suitable for administration
to a patient. In order to administer the emulsion, the composition
may be provided in a container holding the emulsion. The container
may form part of a kit along with instructions for administration
of the composition. Where the administration route is parenteral
such as subcutaneous, intramuscular or intravenous, the container
may be an administration device comprising a pre-determined amount
of the formulation, e.g. a pre-filled syringe.
The container preferably comprises an inert gas added to displace
air and hence maximise storage stability.
Suitable containers may have a volume up to 10 ml, such as 1 to 10
ml.
Treatment
The emulsions of the invention are proposed for use in the
treatment or prevention of inflammatory disorders or proliferative
conditions including psoriasis, glomerulonephritis, lupus
nephritis, diabetic nephropathy, rheumatoid arthritis, dermatitis
and cancers such as skin cancer. In particular, the emulsions of
the invention are proposed for use in the treatment or prevention
of inflammatory disorders or proliferative conditions including
diabetic nephropathy and arthritis such as rheumatoid
arthritis.
By treating or treatment is meant at least one of: (i). inhibiting
the disease i.e. arresting, reducing or delaying the development of
the disease or a relapse thereof or at least one clinical or
subclinical symptom thereof, or (ii). relieving or attenuating one
or more of the clinical or subclinical symptoms of the disease.
By prevention is meant (i) preventing or delaying the appearance of
clinical symptoms of the disease developing in a mammal
The benefit to a subject to be treated is either statistically
significant or at least perceptible to the patient or to the
physician. In general a skilled man can appreciate when "treatment"
occurs. It is particularly preferred if the composition of the
invention are used therapeutically, i.e. to treat a condition which
has manifested rather than prophylactically. It may be that the
composition of the invention is more effective when used
therapeutically than prophylactically.
The composition of the invention can be used on any animal subject,
in particular a mammal and more particularly to a human or an
animal serving as a model for a disease (e.g., mouse, monkey,
etc.).
In order to treat a disease an effective amount of the active
composition needs to be administered to a patient. A
"therapeutically effective amount" means the amount of a
composition that, when administered to an animal for treating a
state, disorder or condition, is sufficient to effect such
treatment. The "therapeutically effective amount" will vary
depending on the composition, the disease and its severity and the
age, weight, physical condition and responsiveness of the subject
to be treated and will be ultimately at the discretion of the
attendant doctor.
It may be that to treat cancer according to the invention that the
composition of the invention has to be readministered at certain
intervals. Suitable dosage regimes can be prescribed by a
physician.
It will be appreciated that pharmaceutical composition for use in
accordance with the present invention is ideally in a form for
parenteral administered, e.g. as an emulsion for injection.
Therapeutic doses will generally be between about 10 and 2000
mg/day and preferably between about 30 and 1500 mg/day. Other
ranges may be used, including, for example, 50-500 mg/day, 50-300
mg/day, 100-200 mg/day.
Administration may be once a day, twice a day, or more often, and
may be decreased during a maintenance phase of the disease or
disorder, e.g. once every second or third day instead of every day
or twice a day. The dose and the administration frequency will
depend on the clinical signs, which confirm maintenance of the
remission phase, with the reduction or absence of at least one or
more preferably more than one clinical signs of the acute phase
known to the person skilled in the art.
The invention is described further below with reference to the
following non-limiting examples.
EXAMPLES
The following compounds are used in the Experiments:
##STR00004##
Example 1
Stability Studies
The formulations were prepared in duplicate for the stability
study:
TABLE-US-00001 TABLE 1a Comparative A/B C/D AY-14 AY15-01 Amount
Amount Amount Amount Component (mg/mL) (mg/mL) (mg/mL) (mg/mL)
Compound B 5 mg 20 mg 2.0 mg 2.0 mg Polysorbate 80 5 mg -- 2.0 mg
2.0 mg MCT Oil 5 mg To make 1 ml 2.0 mg 2.0 mg Citrate buffer To
make 1 ml -- qs to make .sup.a) 1 mL The citrate buffer is composed
of Citric Acid Monohydrate 1.5 mM; Trisodium Citrate dehydrate 8.5
mM and WfI pH = 7, and argon is used for air displacement. .sup.a)
Compound B is solubilized in 10 mM sodium citrate (pH 7) by use of
MCT oil 2 mg/ml and Polysorbate 80, 2 mg/ml; the samples have been
saturated with argon as part of the filling process, and are filled
in 10 ml freeze-stable polymer-vials.
In Table 1b below, samples have been prepared by accurately
weighing out the testing material, ca 500 mg, into 10 ml volumetric
flasks and dilute to volume with ACN/water 95:5. Duplicate samples
are prepared for each time point.
TABLE-US-00002 TABLE 1b Calculation of Compound B content in
AY15-01 samples (duplicate samples). Compound B in Average Weight
sample Compound B Samples (mg) Dilution Area (mg/g*) (mg/g*)
AY15-01-i Start Analysis Sample 1 515.40 10 ml 1534.2 1.73 1.72
Sample 2 523.04 10 ml 1540.4 1.71 AY15-01-j 2 Months Analysis
Sample 1 505.83 10 ml 1504.2 1.62 1.62 Sample 2 509.25 10 ml 1510.6
1.62 AY15-01-k 4 Months Analysis Sample 1 504.93 10 ml 1481.4 1.60
1.60 Sample 2 504.47 10 ml 1486.2 1.61 AY15-01-l 6 Months Analysis
Sample 1 492.74 10 ml 1396.9 1.55 1.55 Sample 2 486.07 10 ml 1388.2
1.56 *The results are reported as mg/g, thus the density of the
solutions has not been taken into account.
General Procedure
Components: 2 mg Compound B/ml nano-emulsion: MCT oil 2 mg/ml and
P80 2 mg/ml and Citrate buffer 10 mM
TABLE-US-00003 1. Compound B 791 mg 2. MCT oil 750 mg 3.
Polysorbate 80 750 mg 4. Sodium citrate buffer 10 mM pH 7to make
375 ml
Components 1 and 2 are mixed at ambient temperature in a vessel and
flushed with argon. Component 3 is added with mixing. The blend is
heated to 80-85.degree. C. in a water bath. Component 4 is added at
60-80.degree. C. slowly with frequent mixing/vortexing. The product
was sterilized by filtration, purged with argon and filled into 10
ml vials. Vials are flushed with argon before closure. Vials were
frozen at -20.degree. C.
Assay, Purity and Impurities:
The method used is a RP HPLC method using UV detection. The
conditions are summarised below:
TABLE-US-00004 TABLE 2 Analytical Column 120 EC-C18 (150 .times.
4.6 mm, 2.7 .mu.m particle size) Flow 1.5 ml/min Detection
wavelength 210 nm Temperature 15.degree. C. Inj. Volume 3 .mu.l
Stop Time 42 min Post Time 3 min Mobile phase A
Water:acetonitrile:formic acid (0.02%):300:700:0.2 Mobile phase B
Acetonitril:formic acid (0.02%):1000:0.2 Diluent Acetonitril/Water
(95:5) Gradient Time (min) Conc. B % 0 0 12 80 20 100
For AY-14, three months data shows no change in the parameters
tested indicating good stability at -20.degree. C.
The stability of formulations A-D and 4 repeats of AY14 is shown in
the Table below
TABLE-US-00005 TABLE 3 Total other Time (months), Sulfoxide Thiol
impurities Comp B area Sample Temperature (C.) area.sup.1
area.sup.2 Impurities.sup.3 (% area) (% area) A 0, rt.sup. 3.5 1 0
3.8 91.8 B 0, rt.sup. 3.5 0.8 0 3.3 92.4 Average 3.5 0.9 0 3.55
92.1 A 1 mth, 5.degree. C. 6.1 0.7 2.1 3 88.1 B 1 mth, 5.degree. C.
6.3 0.6 2 3.1 88 Average 6.2 0.65 2.05 3.05 88.05 A 1 mth,
-20.degree. C. 3.5 1.2 0.7 3.4 91.3 B 1 mth, -20.degree. C. 3.5 1.3
0.6 3.1 91.6 Average 3.5 1.25 0.65 3.25 91.45 C 0, rt.sup. D 0,
rt.sup. Average C 3 mth, 5.degree. C. 9.5 low 2.7 5.3 82.5 D 3 mth,
5.degree. C. 9.4 low 2.7 5.2 82.7 Average 9.45 low 2.7 5.25 82.6 C
3 mth, -20.degree. C. 4.2 0.8 1 4.1 89.9 D 3 mth, -20.degree. C.
4.2 0.9 0.8 3.5 90.6 Average 4.2 0.85 0.9 3.8 90.25 C 6 mth,
5.degree. C. 10.9 low 3.2 2.5 83.4 D 6 mth, 5.degree. C. 10.9 low
3.4 2.9 92.8 Average 10.9 low 3.3 2.7 88.1 C 6 mth, -20.degree. C.
4.2 0.9 1.1 1.8 92 D 6 mth, -20.degree. C. 4.2 0.8 0.8 2.8 91.4
Average 4.2 0.85 0.95 2.3 91.7 AY14-e 3 mth, -20.degree. C. 1.9 LOQ
0.2 4.2 93.8 AY14-f 3 mth, -20.degree. C. 1.8 LOQ 0.2 4.3 93.7
AY14-g 3 mth, -20.degree. C. 1.9 0.4 0.2 4.5 93.2 AY14-h 3 mth,
-20.degree. C. 1.9 0.4 0.1 4.3 93.4 AY15-01-i 0, -20.degree. C.
Sample 1 1.7 0.2 LOQ 4.4 93.7 Sample 2 1.7 0.2 LOQ 4.3 93.8
AY15-01-j 2 mth, -20.degree. C. Sample 1 2.0 0.2 LOQ 5.7 92.1
Sample 2 2.0 0.2 LOQ 6.0 91.8 AY15-01-k 4 mth, -20.degree. C.
Sample 1 2.6 0.3 0.2 3.8 93.1 Sample 2 2.5 0.4 0.2 4.5 92.4
AY15-01-l 6 mth, -20.degree. C. Sample 1 3.0 0.3 N/A 7.6 89.1
Sample 2 3.0 0.4 N/A 6.4 90.2 LOQ low means too low to be seen.
.sup.1RRT 0.57 minutes, .sup.2RRT 1.41 minutes, .sup.3RRT 3.78
minutes. The experiments for AY15-01 samples were perfomed in
duplicate, as can be seen from the sets of data for sample 1 and
sample 2. It should be noted that the chromatograms are manually
integrated and some inaccuracy is inherent in the integration due
to overlapping/tailing peaks and "ghost peaks". However, the amount
of sulfoxide reported should be considered accurate, as no ghost
peaks/overlapping peaks are observed in this retention area. As can
be seen for the above data, the Compound B peak area for
formulations A/B were essentially unchanged even after 6 months of
storage at -20.degree. C.
Example 2
Further formulations have been prepared:
TABLE-US-00006 TABLE 4 AY12 Micelles Ingredient AY12-01 AY12-02
AY12-03 Compound B 1.0 mg 2.0 mg 5.0 mg (100%) Polysorbate 80 5.0
mg 10.0 mg 25.0 mg MCT Oil 5.0 mg 10.0 mg 25.0 mg EDTA 5 mM 5 mM 5
mM Water for qs to make 1 mL qs to make 1 mL qs to make 1 mL
Injection
All three formulations are at pH=7, and argon is used for air
displacement. These examples can be summarised as: AY12-01:
Compound B 1.0 mg/ml IV inj ("Micelles": P80 5 mg/ml MCT oil 5
mg/ml Na-EDTA 5 mM pH 7) AY12-02: Compound B 2.0 mg/ml SC inj.
("Micelles": P80 10 mg/ml MCT oil 10 mg/ml Na-EDTA 5 mM pH 7)
AY12-03: Compound B 5.0 mg/ml SC inj. ("Micelles": P80 25 mg/ml MCT
oil 25 mg/ml Na-EDTA 5 mM pH 7)
These materials are tested against comparative examples: AY12-04:
Compound B 2.0 mg/ml SC inj. (in pure MCT oil) AY12-05: Compound B
5.0 mg/ml SC inj. (in pure MCT oil) AY12-06: Compound B 2.5 mg/ml
IP inj. (in pure DMSO)
The data is reported in table 5
TABLE-US-00007 Time (months), Sulfoxide Thiol Total other Comp B
Sample Temperature (C.) area.sup.1 area.sup.2 Impurities.sup.3
impurities area AY12-01 0 0.7 Not detected LOQ 2.1 97.2 AY12-01 0
0.8 Not detected LOQ 2.4 96.8 AY12-02 0 0.7 Not detected LOQ 2.5
96.9 AY12-02 0 0.7 Not detected LOQ 2.8 96.5 AY12-03 0 0.7 Not
detected LOQ 2.9 96.4 AY12-03 0 0.7 Not detected LOQ 2.5 96.8
AY12-04 0 3.6 Not detected LOQ 2.7 93.8 AY12-04 0 3.6 Not detected
LOQ 2.7 93.7 AY12-05 0 4.3 Not detected LOQ 2.4 93.4 AY12-05 0 4.2
Not detected LOQ 2.3 93.5 AY12-06 0 0.8 Not detected LOQ 3.1 96.1
AY12-06 0 0.8 Not detected LOQ 2.6 96.6 AY12-01 1 mth, 2-8.degree.
C. 1.7 Not detected 0.3 3.2 94.8 AY12-01 1 mth, 2-8.degree. C. 1.7
Not detected 0.3 3.5 94.5 AY12-02 1 mth, 2-8.degree. C. 1.1 0.1 0.2
3.8 94.8 AY12-02 1 mth, 2-8.degree. C. 1.1 0.2 0.2 3.3 95.2 AY12-03
1 mth, 2-8.degree. C. 1.3 0.1 0.2 3.7 94.6 AY12-03 1 mth,
2-8.degree. C. 1.3 0.1 0.2 3.7 94.7 AY12-04 1 mth, 2-8.degree. C.
5.9 0.2 LOQ 3.6 90.3 AY12-04 1 mth, 2-8.degree. C. 5.9 0.3 LOQ 3.8
90.1 AY12-05 1 mth, 2-8.degree. C. 6.8 0.3 LOQ 4.4 88.6 AY12-05 1
mth, 2-8.degree. C. 6.8 0.3 LOQ 4.2 88.7 AY12-06 1 mth, 2-8.degree.
C. 3.2 0.5 LOQ 5.3 91.1 AY12-06 1 mth, 2-8.degree. C. 3.2 0.4 LOQ
5.2 91.2
Example 3
Background
Collagen induced arthritis (CIA) is a commonly used arthritis model
in preclinical research. In this model, the disease is induced by
injecting type II collagen (a cartilage component) emulsified in
complete Freund's adjuvant in the high responder DBA/1 mouse strain
(or in other species such as the rat or the monkey). Introduction
of collagen breaks tolerance and directs an immune-mediated
inflammatory attack on the joints that can be measured by
evaluating standard disease parameters as based on an arthritis
index score: (Animal models for arthritis: innovative tools for
prevention and treatment, Ann Rheum Dis. 2011 August; 70 (8):
1357-62).
Test Article & Formulation
The following compound is used:
##STR00005##
Compound B was formulated for subcutaneous administration in a
nano-emulsion containing 2 mg/ml Compound B, 2 mg/ml MCT oil
(Lipoid), 2 mg/ml Polysorbate 80 (Fluka) and sodium citrate buffer
10 mM (pH 7). The final solution was sterilized by filtration and
saturated with Argon 5.0 AGA prior to aliquoting in 10 ml tubes
stored at -20.degree. C. till further use.
Animals
7-10 weeks old male DBA/1 mice were sourced from Beijing Vital
River laboratories, China (average weight at the beginning of the
study: 18-25 g). The animals were housed in an animal room set at a
temperature of 23.+-.2.degree. C., humidity of 40-70%, under a
12-hour light/12-hour dark cycle. SPF (Serum Pathogen Free) mouse
growth breeding feed (Beijing KeaoXieli feed Co. LTD) was provided
ad libitum throughout the in-life portion of the study. Water was
available ad libitum.
Collagen Induced Arthritis (CIA)--Experimental Design
Groups
40 male DBA/1 mice were divided into 4 groups and treated according
to the design below.
TABLE-US-00008 TABLE 6 Dosing Dosing Dosing Animals Group Treatment
route schedule volume number 1 Sham (Non-treatment) -- Daily 2
ml/kg 7 2 Vehicle (DMSO) Intraperitoneal Daily 2 ml/kg 11 3
Compound B 5 mg/kg Subcutaneous Daily 2.5 ml/kg 11 4 Compound B 10
mg/kg Subcutaneous Daily 5 ml/kg 11
Induction of CIA
CIA was induced in male DBA/1 mice (except sham group) by
immunization with 0.1 ml emulsion containing an equal volume of
bovine type II collagen solution (2 mg/me and Freund's complete
adjuvant (FCA) at the tail base. The first injection was given on
Day 0 and the second injection as booster was given on Day 21.
Treatment started one hour before the second collagen injection and
continued for at least 21 days.
CIA Assessment and Treatment
CIA was assessed in mice by two blinded observers to measure paw
swelling with a capacity measurement method. The occurrence of
arthritis was observed by scoring all four paws for severity of
erythema and swelling, using a clinical score ranging from 0 (no
swelling) to 4 (severe swelling and erythema) as below, yielding a
maximum Arthritis Index (AI) score of 16 (4 paws.times.max score
per paw): J Med Chem. 2014 Sep. 25; 57 (18):7523-35, Kokotos et
al.
Score Grading:
TABLE-US-00009 Grade Degree 0 No swelling, normal appearance 1
Slight swelling and erythema of the ankle, wrist or digits 2
Moderate swelling and erythema of the ankle, wrist or digits 3
Severe swelling and erythema of the ankle, wrist or digits 4
Maximal inflammation with joint rigidity; very severe
The detailed dosing info per treatment group was as follows.
Group 1: 7 mice that have received no collagen, treated with
vehicle DMSO (everyday) for 3 weeks.
Group 2: 11 mice were induced by collagen and treated with vehicle
DMSO (everyday) for 3 weeks.
Group 3: 11 mice were induced by collagen and treated with Compound
B (5 mg/kg, Daily) for 3 weeks.
Group 4: 11 mice were induced by collagen and treated with Compound
B (10 mg/kg, Daily) for 3 weeks.
At the end of the study, all mice were euthanized according to
applicable ethical laws by carbon dioxide inhalation.
Clinical Observations
The physical condition of the animals and clinical signs (if any)
were recorded for individual animals, once before commencement of
treatment and once daily during the study. Observation was
performed at the same time interval each day. Body weigh
measurements were also conducted on the day of allocation to its
corresponding group and then on each day of arthritis
evaluation.
Statistical Analysis
Data was given as Mean.+-.SD. The statistical analysis of the
results were performed by the Independent-Sample t test. Values for
p<0.05 were considered significant.
Results
Effect of Compound B on Body Weight in the CIA Mouse Model
From Day 32 to the end of the experiment a significant reduction in
body weight was observed in the CIA control mice, compared with the
sham mice (p<0.005). Such weight loss or wasting is typical of
this model as CIA progresses. There was a significant inhibition of
the body weight loss of Compound B 5 mg/kg group on Day 34 and
39.about.43 (p<0.05.about.0.005) and Compound B 10 mg/kg group
on Day 25 and 32.about.43 (p<0.05.about.0.005), compared with
vehicle treated CIA group (Table 7).
Effect of Compound B on Arthritis Index (AI) in the CIA Mouse
Model
A 100% incidence of CIA was observed by day 32 in CII-immunized
mice, with a maximum AI of 4.18 observed at Day 43 post
immunization. The AI and incidence of all groups increased in a
time-dependent mode from day 27 to day 43.
The AI of Compound B 10 mg/kg group on Day 41.about.43 was
significantly reduced (p<0.05) in comparison to the CIA control
group. The AI index of Compound B 5 mg/kg group was also reduced
when compared with that of the CIA control group, however, the
reduction reached no statistical significance (p>0.05) (Table
8).
No other clinical observations or side effects were evident in
animals treated with Compound B.
The study results indicated, among other things, that arthritis was
successfully induced in DBA/1 mice. Treatment with subcutaneously
administered. Compound B reduced arthritis symptoms and disease
score in the absence of side effects.
TABLE-US-00010 TABLE 7 Compound B on body weight in the CIA mouse
model Day Dose Group (mg/kg) -1 20 25 27 29 32 34 36 39 41 43 Sham
-- 21.57 .+-. 23.17 .+-. 21.8 .+-. 21.59 .+-. 22.14 .+-. 22.4 .+-.
22.4 .+-. 22.66 .+-. 22.34 .+-. 22.04 .+-. 22.41 .+-. 1.93 2.01
2.17 2.15 2.28 2.1 *** 2.04 *** 2.14 *** 2.11 *** 1.91 *** 2.03 ***
Vehicle -- 21.62 .+-. 22.16 .+-. 20.48 .+-. 20.6 .+-. 20.55 .+-.
19.36 .+-. 19.17 .+-. 19.52 .+-. 19.16 .+-. 19.27 .+-. 19.46 .+-.
1.06 1.14 1.19 1.28 1.67 1.48 1.41 1.33 1.23 1.36 1.4 Cpd B 5 21.55
.+-. 22.5 .+-. 21.53 .+-. 21.06 .+-. 20.85 .+-. 20.25 .+-. 20.55
.+-. 20.63 .+-. 20.59 .+-. 21.09 .+-. 21.39 .+-. 1.19 1.27 1.18
1.37 1.28 1.41 1.25 * 1.27 1.15 * 1.24 *** 1.01 *** Cpd B 10 22.12
.+-. 22.3 .+-. 21.69 .+-. 21.37 .+-. 21.61 .+-. 21.38 .+-. 21.46
.+-. 21.81 .+-. 21.7 .+-. 21.95 .+-. 22.35 .+-. 1.37 1.44 1.4 * 1.5
1.75 2.04 * 2.1 ** 2.25 ** 1.9 *** 1.76 *** 1.59 *** * p < 0.05,
** p < 0.01, *** p < 0.005 vs CIA control group
TABLE-US-00011 TABLE 8 Effect of Compound B on arthritis index (AI)
in the CIA mouse model Day Dose Group (mg/kg) -1 20 25 27 29 32 34
36 39 41 43 Sham -- 0 .+-. 0 .+-. 0 .+-. 0 .+-. 0 .+-. 0 .+-. 0
.+-. 0 .+-. 0 .+-. 0 .+-. 0 .+-. 0 0 0 0 0 * 0 *** 0 *** 0 *** 0
*** 0 *** 0 *** Vehicle -- 0 .+-. 0 .+-. 0 .+-. 0.36 .+-. 1 .+-.
2.73 .+-. 3.82 .+-. 4 .+-. 4.09 .+-. 4.09 .+-. 4.18 .+-. 0 0 0 0.67
1 1.49 1.66 1.9 1.81 1.81 1.94 Cpd B 5 0 .+-. 0 .+-. 0 .+-. 0.36
.+-. 1.27 .+-. 3 .+-. 3.27 .+-. 3.36 .+-. 3.36 .+-. 3 .+-. 2.91
.+-. 0 0 0 0.67 1.19 1.79 2.33 2.42 2.58 2.49 2.34 Cpd B 10 0 .+-.
0 .+-. 0 .+-. 0.18 .+-. 0.55 .+-. 1.36 .+-. 2 .+-. 2.18 .+-. 2.27
.+-. 2.09 .+-. 2.09 .+-. 0 0 0 0.6 1.51 1.86 2.97 2.96 2.9 2.55 *
2.30 * * p < 0.05 **p < 0.01, *** p < 0.005 vs CIA control
group
Example 4
The following compound is used:
##STR00006##
The STZ diabetic nephropathy model is frequently used in
preclinical diabetic & kidney research as it recapitulates
several features of the human disease. In this model,
streptozotocin (STZ), a glucosemoiety with a very reactive
nitrosourea group from the mould Streptomyces griseus, is injected
in rats (or mice) to induce selective destruction of the
insulin-producing .beta.-cells of the pancreas. This results in
increasing blood glucose levels which within a period of 4 to 6
weeks (and extending up to 24 weeks) lead to progressive kidney
disease characterized by altered renal haemodynamics, increased
glomerular basement membrane thickness and promotion of the
development of inflammation and fibrosis in the kidney.
Importantly, disease progression in this model can be monitored by
analysing increasing levels of various biochemical (urine protein,
albumin, protein to creatinine rations etc) or histological markers
(a-SMA, Fibronectin etc) allowing direct assessment of action of
chronically administered agents, Tesch, Nephrology (Carlton). 2007
June; 12 (3):261-6.
Test Article & Formulations
Compound B was formulated for subcutaneous administration in a
nano-emulsion containing 2 mg/ml Compound B, 2 mg/ml MCT oil
(Lipoid), 2 mg/ml Polysorbate 80 (Fluka) and sodium citrate buffer
10 mM (pH 7). The final solution was sterilized by filtration and
saturated with Argon 5.0 AGA prior to aliquoting in 10 ml tubes
stored at -20.degree. C. till further use. A separate Polysorbate
80- and sodium citrate buffer-based vehicle solution without
Compound B and MCT oil was also prepared, sterilized, aliquoted and
stored, serving as the subcutaneous vehicle control used in the STZ
animal study.
Animals
5-6 weeks old male Sprague-Dawley (SD) rats were sourced from
Harlan Laboratories, Houston, Tex., USA (average weight at the
beginning of the study: 125-135 g). The animals were housed in an
animal room set at a temperature of 23.+-.2.degree. C., humidity of
40-70%, under a 12-hour light/12-hour dark cycle. Labdiet 5001 (PMI
Nutrition International) was provided ad libitum throughout the
in-life portion of the study. Water was available ad libitum.
Experimental Instruments and Assays
The following instruments were used:
Chemistries:
Blood glucose: ReliOn Ultima Glucometer & test strips
Urine protein: Bradford assay kit: Quick Start.COPYRGT. Bradford
dye reagent and BSA standard set. Catalog #500-0205 (Bio-Rad
Laboratories, Inc. Hercules, Calif., USA). Quick Start is a
colorimetric assay based on the reaction of proteins with an
alkaline copper tartrate solution and Folin reagent.
Urine Albumin: Nephrat II Quantitative Determination of Rat Urinary
Albumin Kit. Catalog #NR002 Strip Plate (Exocell Inc.,
Philadelphia, Pa., USA). Nephrat II is a direct competitive ELISA
for the quantitative determination of urinary albumin in rat
specimens. An anti-rat albumin antibody-HRP conjugate is allowed to
react with standards and diluted urine samples. A chromogenic
substrate, TMB, is added to the wells, and color development
ensues. Color intensity is inversely proportional to the log of rat
albumin concentration of the sample.
Urine creatinine: The Creatinine Companion Assay Kit. Catalog #1012
(Exocell, Inc., Philadelphia, Pa., USA) was used to measure urine
creatinine. The procedure is an adaptation of the alkaline picrate
method and entails determination of the differential absorbance in
a sample before and after the additions of acid to correct for
color generation due to interfering substances.
Assay instrument: Bio-Rad iMark microplate reader.
Immunohistochemistry:
ImmPRESS HRP Anti-Mouse IgG, Rat adsorbed (Peroxidase) Polymer
Detection Kit, MP-7422, Vector Laboratories, Inc. Burlingame,
Calif.
Quenching Endogenous Peroxidase Activity: 0.3% H.sub.2O.sub.2
Peroxidase (HRP) Substrate Kit, 3,3'-diaminobenzidine Kit,
SK-4100
Histopathological analysis was conducted using an Olympus BX51
research microscope equipped with a DP-71 digital camera. Image-Pro
Plus software was used for computer-assisted image analysis.
Groups
50 male Sprague-Dawley SD rats were divided into 5 groups of 10
rats and treated according to the design below.
TABLE-US-00012 TABLE 9 Groups and Treatments Dosing Dosing Group
Description route volume Animals No. 1 Sham (vehicle) sc, daily
0.67-1.0 ml 10 2 STZ control (vehicle) sc, daily 0.67-1.0 ml 10 3
STZ + Compound B sc, daily 0.67-1.0 ml 10 [2 mg/kg] 4 STZ +
Compound B sc, daily 0.67-1.0 ml 10 [4 mg/kg] 5 STZ + Compound B
sc, daily 0.67-1.0 ml 10 [6 mg/kg]
Induction of Diabetic Nephropathy (DN)
DN was induced in rats (except sham group--Group 1) by tail vein
injection of streptozotocin (STZ) (S0130, Sigma Aldrich
Corporation, St. Louis, Mo. USA) at a dose of 50 mg/kg in 0.4-0.45
ml of citrate buffer. Sham control received a similar injection in
citrate buffer in the absence of STZ. The injection was given on
Day 0. Treatment started on Day 3 and continued for up to 6
weeks.
Group Treatments
Table 9 above summarizes the treatment schedules of the study.
Overall, 3 different doses (2, 4 and 6 mg/kg) of Compound B were
administered subcutaneously (SC) for 6 weeks following induction of
diabetes. Vehicle control was administered in Group 1 (animals that
did not receive STZ) and Group 2 (animals that have received STZ),
daily for 6 weeks. Periodical collection of urine and blood was
performed to allow multiple biochemical analyses as described
below.
Group Assessments
The following assessments were performed on animals of all groups
throughout the course of this study:
Clinical signs: All clinical signs were recorded for individual
animals, once before commencement of treatment and once daily
during the study. Observation was performed at the same time
interval each day.
Body weight: Each animal was weighed on the day of allocation to
its corresponding group and then every time prior to injection of
each test article for dose volume adjustments.
Sample Collection & Assessments
Clinical Chemistry Assessments
Blood Glucose: Blood glucose was measured at the 0, 1, 2, 4, and 6
week time points by collecting a drop of blood from freshly pierced
tip of the tail using a sterile 25 G needle. Blood glucose
measurements were conducted immediately by direct test strip
analysis.
Urine Protein: At the 0, 2, 3, 4, 5 and 6 week time points the rats
were set up in metabolic cages for urine collection. Measurements
of urine protein were performed on 12 hour void volumes ranging
from 7-16 ml. All 12 hour urine protein values (expressed in mg)
were extrapolated to 24 hours to correspond to standard practice in
the field.
Urine Albumin: Measurements of urine albumin were performed on
samples collected at the 0 and 6 week time points. As with urine
protein data the albumin values were extrapolated to 24 hours to
correspond to standard practice in the field.
Urine Creatinine: Measurements of urine creatinine were performed
on samples collected at the 0 and 6 week time points. The urine
creatinine data was used to determine protein and albumin ratios
and expressed as milligram Creatinine: gram protein or albumin
(mg/g) to correspond to standard practice in the field.
Terminal Studies
Euthanasia: Animals that have completed the scheduled test period
were disposed with carbon dioxide according to standard ethical
guidelines. All animals were subjected to necropsy, supervised by a
pathologist, as detailed below. The scores of sacrificed animals
were included in the calculations of the mean group scores
throughout the experiment and until its end.
Necropsy: On the completion day, sacrifices were performed at
approximately 5 hours after the last application. A macroscopic
examination of the animal was performed on all sacrificed animals
and any abnormality was recorded.
Kidney tissue collection: Immediately after the blood collection,
the left and right kidneys were excised, cut in 3-4 mm thick
coronal slices. A central slice of kidney was placed in formalin
for subsequent embedment and routine histological assessments.
Several slices were placed on aluminum foil strips and flash frozen
by immersion in liquid nitrogen and placed in air-tight storage
vials for subsequent immunohistochemical assessment. All samples
were frozen at -80.degree. C. until analyzed. The following
analyses were performed:
Histopathological Analysis
Glomerular .alpha.-SMA staining (for area of glomerular mesangial
activation): Frozen kidney sections were cut and batch stained for
.alpha.-SMA using a monoclonal primary antibody (Clone 1A4, Sigma
Chemical Co, St Louis, Mo.) using standard immunohistochemical
methods. Peroxidase-conjugated second antibody was detected
utilizing ImmPress polymer detection reagents according to the
manufacturer's instructions (Vector Laboratories, Inc. Burlingame,
Calif.). Group 3 (STZ+Compound B [2 mg/kg] sc) was omitted from the
analysis.
Glomerular fibronectin staining (for area of glomerular matrix):
Frozen kidney sections were cut and batch stained for fibronectin
staining using a monoclonal primary antibody (Clone IST-9) AbCam
(Cambridge, Mass.) and standard immunohistochemical methods
conducted at Probetex (See attached SOP). Group 3 (STZ+Compound B
[2 mg/kg] sc) was omitted from the analysis.
Computer Assisted Image Analysis
.alpha.-SMA and Fibronectin Staining
.alpha.-SMA: (area of mesangial cell activation): Glomerular
.alpha.-SMA staining was assessed by image analysis. Digital images
were taken of twenty-five random glomeruli at an objective
magnification of 10.times.. Total area of glomerular staining was
measured by computer assisted image analysis using the tracing tool
of Image-Pro imaging software to capture glomerular area. The
images were segmented and converted to gray scale then
pseudo-colored representing background (blue) and positive staining
(red) areas and percent area representing .alpha.-SMA positive
mesangial cells was quantitated. Data were saved on an excel
spreadsheets and average staining area was calculated and analyzed
statistically.
Fibronectin: (area of mesangial cell matrix accumulation): Image
analysis of glomerular staining for fibronectin was conducted using
the same methods discussed above for .alpha.-SMA.
Results--Clinical
Blood glucose: Average blood glucose at start of the study was 127
mg/dl (range 120-131 mg/dl). All rats receiving STZ became diabetic
(blood glucose >200 mg/dl) at the start of the experiment,
except for rats #11 (Group 2), 21, 31 and 32 (Group 4), and 41
(Group 5). Of these rats, only #11 progressed to values greater
than 200 mg/dl for the remainder of the study. Most remaining rats
developed hyperglycemia by the 2 week time point. However, despite
elevated glucose readings at the start of the 2 week period, some
of the rats (distributed among the groups) did not sustain their
diabetic status. All remaining animals at 4 and 6 weeks had glucose
values above 200 mg/dl. The sensitivity of the meter measures a
maximum of 500 mg/dl, thus some of these values may be under
estimates. Average blood glucose levels for each group are
illustrated in the summary table below.
TABLE-US-00013 TABLE 10 Blood Glucose Measurements Group 0 wk 3
days 6 days 2 wks 4 wks 6 wks G1: Critrate (iv) + Vehicle, (sc,
daily) 129.8 111.6 111.8 122.0 112.7 110.3 G2: STZ (iv) + Vehicle,
(sc, daily) 130.0 288.6 362.7 359.6 311.8 293.0 G3: STZ + Compound
B [2 mg/kg] 129.6 300.3 339.5 340.1 239.5 210.8 sc, daily G4: STZ +
Compound B [4 mg/kg] 120.3 285.5 349.8 327.4 272.0 251.0 sc, daily
G5: STZ + Compound B [6 mg/kg] 129.0 305.1 372.8 370.1 321.8 304.3
sc, daily
Urine Protein: Average pretrial urine protein values ranged from
0.7 to 3.7 mg/dl among the groups. STZ controls (Group 2), showed
an initial spike in proteinuria at 2 weeks with 43.5 mg/24 hrs.
These values lessened thereafter, but remained elevated at 27.2,
33.9, 30.2 and 34.2 mg/24 hours at 3, 4, 5, and 6 weeks,
respectively. Proteinuria in vehicle controls (Group 1) ranged from
17.0-24.6 mg/24 hours throughout the course of the study. Compound
B treatment resulted in a reduction in proteinuria relative to
STZ+vehicle as indicated in Table 11 below.
Urine protein-to-creatinine ratios provide more accurate estimates
of the urinary protein excretion rate and are not affected by
hydration. Thus, urine protein to urine creatinine ratios were
determined at 0 and 6 week time points. The results showed similar
trends as protein measurements alone, however differences among the
groups were more defined. For example, STZ+vehicle showed a 70%
increase in UP/CR (1715: 2919) relative to citrate control at the 6
week time point. All treatment groups (except low-dose Compound B)
had ratios less than STZ+vehicle; reductions followed a
dose-dependent pattern.
TABLE-US-00014 TABLE 11 Proteinuria & Protein/Creatinine Ratio
(0 time to 6 weeks). Proteinuria (mg/24 hr) UP/CR mg/g Group 0 wk 2
wks 3 wks 4 wks 5 wks 6 wks 0 wk 6 wks G1: Critrate (iv) + Vehicle,
(sc, daily) 0.8 24.6 17.0 20.8 20.5 21.9 140.5 1714.9 G2: STZ (iv)
+ Vehicle, (sc, daily) 2.7 43.5 27.2 33.9 30.2 34.2 497.9 2918.7
G3: STZ + Compound B [2 mg/kg] 1.5 30.8 30.4 39.4 39.1 40.0 239.6
3159.0 sc, daily G4: STZ + Compound B [4 mg/kg] 1.1 18.8 19.0 24.5
25.5 27.7 165.5 2384.5 sc, daily G5: STZ + Compound B [6 mg/kg] 3.7
21.4 21.7 22.5 27.1 25.8 650.0 1903.7 sc, daily
Urine Albumin: Urine albuminuria is a more sensitive measurement of
glomerular permeability defects than urine protein, particularly in
diabetic nephropathy. As with urine protein-to-creatinine,
albumin-to-creatinine ratios provide more accurate estimates of the
urinary albumin excretion rate, and are not affected by hydration.
Thus, urine albumin and urine albumin:creatinine ratios were
measured at 0 and 6 week time points. The results showed similar
trends observed for urine protein data where baseline average
pretrial urine albumin:creatinine ratios ranged from 128 to 387
among the groups. Albumin:creatinine ratio in citrate controls
(Group 1) averaged 457 at 6 weeks of diabetes. The highest
albumin:creatinine ratio was observed in the STZ+vehicle (Group 2)
which increased to 1584 at 6 weeks of diabetes. All groups of rats
treated with STZ+Compound B had albumin:creatinine ratios less than
STZ+vehicle (Table 12).
TABLE-US-00015 TABLE 12 Urine Albumin & Urine
Albumin/Creatinine Ratio. Urine Albumin & UA/Creatine Ratio
mg/24 hr UA/CR mg/g Group 0 wk 6 wks 0 wk 6 wks G1: Critrate (iv) +
Vehicle, (sc, daily) 1.3 5.6 247.2 456.6 G2: STZ (iv) + Vehicle,
(sc, daily) 2.3 18.5 386.7 1583.6 G3: STZ + Compound B [2 mg/kg]
0.8 18.8 128.5 1492.4 sc, daily G4: STZ + Compound B [4 mg/kg] 0.8
10.9 127.8 942.9 sc, daily G5: STZ + Compound B [6 mg/kg] 1.7 13.9
302.3 1011.9 sc, daily
Body Weights, Toxicity of Compounds, and Mortality
Sham-vehicle (Group 1) rats progressively gained weight as
expected. Growth of all animals treated with STZ was stunted
relative to sham based on average body weight measurements for each
group. The health of the animals throughout the course of the study
appeared very good and there were no toxicities or mortality
observed for Compound B treated groups.
Results--Histopathology
Glomerular .alpha.-SMA Staining (Mesangial Cell Activation)
Basal glomerular mesangial cell a-SMA staining is enhanced upon
activation in a number of renal diseases including diabetic
nephropathy Sham controls (Group 1) had the least mesangial a-SMA
where average areas of staining were 2.1% of the glomerular area.
Vehicle-treated STZ controls (Group 2) showed the highest level of
mesangial .alpha.-SMA staining reaching 4.6% of the glomerular
area. Treatment with Compound B blunted STZ related mesangial cell
activation and a-SMA staining in all treatment groups was less than
STZ-vehicle controls (Table 13).
TABLE-US-00016 TABLE 13 Glomerular .alpha.-SMA staining (percentage
of staining per field) a-SMA staining Group 1 Group 2 Group 4 Group
5 Mean 2.10 4.63 3.57 3.21 SD 0.95 1.76 1.21 1.16
Glomerular Fibronectin Staining (Mesangial Matrix Expansion)
Glomerular fibronectin staining, used as an index of matrix
accumulation, showed similar trends as observed for a-SMA. As with
a-SMA, glomerular mesangial Fn staining is enhanced upon activation
in renal disease including diabetic nephropathy. Basal Fn
expression in sham controls (Group 1) averaged 18.2% of the
glomerular area. Vehicle-treated STZ controls (Group 2) showed high
levels of mesangial Fn staining reaching 22.8% of the glomerular
area. Treatment with Compound B resulted in a significant reduction
in Fn expression relative to vehicle controls (Table 14).
TABLE-US-00017 TABLE 14 Glomerular FN staining (percentage of
staining per field) FN staining Group 1 Group 2 Group 4 Group 5
Mean 18.2 22.8 13.1 15.3 SD 2.9 4.3 4.5 3.1
Streptozotocin (STZ) induced diabetes and diabetic nephropathy
typical of this model as indicated by increased blood glucose,
proteinuria, albuminuria, and histopathological changes
characterized by glomerular mesangial cell activation (assessed by
acquisition of alpha-smooth muscle actin, .alpha.-SMA) and
accumulation of matrix protein (fibronectin) relative to sham
controls. Compound B, administered in a subcutaneous formulation,
was effective in reducing proteinuria, albuminuria, urine
protein:creatinine and urine albumin:creatinine ratios in this
model (especially at the high dose). Similarly, Compound B reduced
glomerular expression of glomerular .alpha.-SMA and Fn-protein
expression assessed by immunohistochemical evaluation. Overall,
Compound B did not induce side effects during a 6-week course of
treatment.
* * * * *
References